CN111004245B

CN111004245B - Pyrazole-pyrimido imidazole compound, preparation method and application thereof

Info

Publication number: CN111004245B
Application number: CN201911269082.9A
Authority: CN
Inventors: 郑友广; 孟龙; 裴鑫; 姜萍; 张务奇; 安琳; 黄统辉
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2019-12-11
Filing date: 2019-12-11
Publication date: 2022-01-07
Anticipated expiration: 2039-12-11
Also published as: CN111004245A

Abstract

The invention relates to a pyrazole-pyrimido imidazole compound, a preparation method and application thereof, belonging to the field of pharmaceutical chemistry and pharmacotherapeutics. The invention provides application of a compound shown in a formula I or pharmaceutically acceptable salt thereof in preparing medicaments for treating tumor-related diseases.

Description

Pyrazole-pyrimido imidazole compound, preparation method and application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry and pharmacotherapeutics, and particularly relates to a pyrazole-pyrimido imidazole compound. The invention also relates to a preparation method of the compounds and a pharmaceutical composition containing the compounds.

Background

As is well known, tumors are one of the diseases with extremely high morbidity and mortality, and the common treatment modes mainly comprise surgical excision, chemotherapy, radiotherapy and some comprehensive measures. But these treatment modalities will undoubtedly have a more or less impact on the quality of life of the patient after healing. Surgical resection does not prevent recurrence and metastasis of the tumor; although various tumors can be radically treated by radioactive therapy, certain limitations exist, such as long treatment period; chemotherapy, as a systemic treatment method, can kill tumor cells in a patient to a greater extent, but has poor selectivity so as to generate systemic side effects and limit the clinical application of the chemotherapy, so that the improvement of the curative effect of chemotherapeutic drugs has great significance for tumor treatment.

Many anti-tumor drugs are already on the market, which bring good news to cancer patients, but the malignant tumor is still a serious disease which is difficult to overcome so far. In our country, the incidence of tumors is rising year by year, increasing at a rate of 10% per year, with 6 diagnosed as malignant tumors and 5 dying from cancer per minute. Moreover, the number of cancer diseases in China accounts for about one fifth of the diseases in the world; cancer deaths account for approximately one-fourth of the total number worldwide. The most common cancers in China are liver cancer (65.3 thousands of patients suffering from the breast cancer in 2014), gastric cancer (40.5 thousands), liver cancer (39.5 thousands) and colorectal cancer (25.3 thousands), and in addition, the number of patients suffering from the breast cancer in female groups is more than 18.7 thousands of patients.

In summary, although there are many kinds of antitumor drugs in clinic, the existing drugs still cannot meet the treatment requirements due to the defects of the antitumor drugs, such as easy generation of drug resistance and great side effects, and the novel antitumor drugs are urgently needed.

Disclosure of Invention

The invention aims to provide a pyrazole-pyrimido imidazole compound which has better inhibitory activity on human chronic myelogenous leukemia cells K562 and human colon cancer cells HCT116 on the basis of the prior art.

Another object of the present invention is to provide a process for the preparation of the above compound.

The third object of the present invention is to provide a pharmaceutical use of the above compound.

The technical scheme of the invention is as follows:

a compound, isomer, or pharmaceutically acceptable salt thereof, represented by formula I,

wherein the content of the first and second substances,

R¹represents C₁-C₆Alkyl, substituted C₁-C₆Alkyl radical, C₁-C₆Alkoxy, cyano, hydroxy, nitro or halogen, said substituted C₁-C₆The alkyl group may be optionally mono-or polysubstituted with the following substituents: hydroxy, nitro or halogen;

R²represents phenyl, substituted phenyl, benzyl, substituted benzyl, C₁-C₆Alkyl, substituted C₁-C₆Alkyl, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopropyl, cyclobutyl, cyclopentyl, or cyclopentylSubstituted cyclopentyl, cyclohexyl or substituted cyclohexyl, and the substituted phenyl, substituted benzyl and substituted C₁-C₆The alkyl, substituted cyclopropyl, substituted cyclobutyl, substituted cyclopentyl or substituted cyclohexyl group may be optionally mono-or polysubstituted with the following substituents: c₁-C₄Alkoxy, cyano, hydroxy, nitro or halogen.

In a preferred embodiment, R¹Represents C₁-C₄Alkyl, substituted C₁-C₄Alkyl radical, C₁-C₄Alkoxy, cyano, hydroxy, nitro, fluoro or bromo, said substituted C₁-C₄The alkyl group may be optionally mono-or polysubstituted with the following substituents: hydroxy, nitro, fluoro, chloro or bromo.

In a more preferred embodiment, R¹Represents methyl, ethyl, methoxy, ethoxy, fluorine, chlorine or bromine. For example, R¹Represents methyl, fluorine or bromine.

In a preferred embodiment, R mentioned in the present invention²Represents phenyl, substituted phenyl, cyclopropyl, substituted cyclopropyl, cyclopentyl or substituted cyclopentyl, which may be optionally mono-or polysubstituted with: methoxy, ethoxy, cyano, hydroxy, nitro or fluoro, chloro or bromo.

In a more preferred embodiment, R²Represents phenyl, substituted phenyl, cyclopropyl, cyclopentyl, said substituted phenyl being optionally mono-or polysubstituted with the following substituents: methoxy, ethoxy, cyano, hydroxy, nitro, fluoro, chloro or bromo.

In a particularly preferred embodiment, R²Represents phenyl or substituted phenyl, said substituted phenyl being optionally mono-or polysubstituted with the following substituents: methoxy, ethoxy, nitro, fluoro, chloro or bromo.

Further, in the compound of formula I or a pharmaceutically acceptable salt thereof, the compound is selected from the following compounds:

still further, the compound of formula I is further selected from the following compounds:

the invention discloses a preparation method of a compound shown in a general formula I, which takes 4-nitropyrazole-3-formic acid and substituted 2-aminoacetophenone as raw materials to obtain a target compound through the reaction steps of esterification reaction, aminolysis reaction, amide dehydration reaction, reduction, delapine reaction and the like, and the specific synthetic route is as follows:

the invention further discloses a preparation method of the more specific compound shown in the general formula I:

the intermediates or the target compounds mentioned in the present invention can be purified according to conventional isolation techniques and converted into addition salts with pharmaceutically acceptable acids as required.

Unless otherwise indicated, the following terms used in the specification and claims have the meanings discussed below:

"pharmaceutically acceptable salts" refers to those salts that retain the biological effectiveness and properties of the parent compound. Such salts include:

(1) salifying with an acid by reaction of the free base of the parent compound with an inorganic or organic acid, including hydrochloric, hydrobromic, nitric, phosphoric, metaphosphoric, sulfuric, sulfurous, perchloric, and the like, the organic acid includes acetic acid, trifluoroacetic acid, propionic acid, acrylic acid, caproic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, oxalic acid, (D) or (L) malic acid, fumaric acid, maleic acid, ascorbic acid, camphoric acid, benzoic acid, hydroxybenzoic acid, γ -hydroxybutyric acid, methoxybenzoic acid, phthalic acid, methanesulfonic acid, ethanesulfonic acid, naphthalene-1-sulfonic acid, naphthalene-2-sulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, lactic acid, cinnamic acid, dodecylsulfuric acid, gluconic acid, glutamic acid, aspartic acid, stearic acid, mandelic acid, succinic acid, glutaric acid, or malonic acid, and the like.

(2) The acidic proton present in the parent compound is replaced by a metal ion such as an alkali metal ion, an alkaline earth metal ion or an aluminum ion, or is complexed with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, quinine, or the like.

"pharmaceutical composition" refers to the combination of one or more of the compounds of the present invention, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, with another chemical ingredient, such as a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to facilitate the administration process to an animal.

"pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" refers to inactive ingredients in a pharmaceutical composition that do not cause significant irritation to an organism and do not interfere with the biological activity and properties of the administered compound, such as, but not limited to: calcium carbonate, calcium phosphate, various sugars (e.g., lactose, mannitol, etc.), starch, cyclodextrin, magnesium stearate, cellulose, magnesium carbonate, acrylic or methacrylic polymers, gelatin, water, polyethylene glycol, propylene glycol, ethylene glycol, castor oil or hydrogenated or polyethoxylated hydrogenated castor oil, sesame oil, corn oil, peanut oil, and the like.

"alkyl" means a saturated aliphatic radical of 1 to 20 carbon atoms, including straight and branched chain radicals (a numerical range referred to herein, e.g., "1 to 20", means that the radical, in this case alkyl, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms). More preferably, the alkyl group is a medium size alkyl group having 1 to 10 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, and the like. Preferably, alkyl is lower alkyl having 1 to 8 or 1 to 6 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl or tert-butyl, and the like. Alkyl groups may be substituted or unsubstituted. When substituted alkyl, the substituent is preferably one or more, more preferably 1 to 3, most preferably 1 or 2 substituents.

"hydroxy" means an-OH group.

"nitro" means-NO₂A group.

"cyano" means a-CN group.

"alkoxy" means-O- (unsubstituted alkyl) and-O- (unsubstituted cycloalkyl). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, and the like.

"halogen" means fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.

"haloalkyl" denotes halogen-substituted alkyl, preferably halogen-substituted lower alkyl as defined above, which is substituted by one or more identical or different halogen atoms, e.g. -CH₂Cl、-CF₃、-CH₂CF₃、-CH₂CCl₃And the like.

The invention provides a pharmaceutical composition, which takes the compound or the pharmaceutically acceptable salt thereof as an active ingredient or a main active ingredient, and is assisted by a pharmaceutically acceptable carrier.

The pyrazole-pyrimido imidazole compound provided by the invention can be applied to the preparation of medicines for treating tumor-related diseases.

By adopting the technical scheme of the invention, the advantages are as follows:

the pyrazole-pyrimido imidazole compound provided by the invention has good inhibitory activity on human chronic myelogenous leukemia cell K562 and human colon cancer cell HCT116, and can be applied to the preparation of medicaments for treating diseases related to tumors.

Detailed Description

The pyrazole-pyrimidoimidazoles compounds of the present invention are further illustrated by the following examples, which are not intended to limit the invention in any way.

EXAMPLE 1 Synthesis of Compound 1

Adding 4-nitropyrazole-3-formic acid (20g,127mmol) into a 500mL three-necked flask, dissolving with 300mL ethanol, slowly dropwise adding thionyl chloride (11mL,140mmol) at 0 ℃ by using a constant-pressure dropping funnel, controlling the internal temperature to be less than 5 ℃ in the whole process, and after the addition, heating the reaction solution to room temperature and stirring for 20 hours. After the reaction was completed, the solvent was evaporated by a rotary evaporator and dried to obtain 22.8g of a white solid with a yield of 97%, TLC (PE: EA ═ 1:1,1d acetic acid v/v).

EXAMPLE 2 Synthesis of Compound 2

Compound 1(10g,54mmol) was added to a 250mL pressure bottle, dissolved in 150mL excess ammonia, and heated to 100 ℃ in an oil bath and refluxed for 5 h. After the reaction, the reaction mixture was naturally cooled to room temperature, a white solid was precipitated, and the precipitate was filtered under suction to obtain a cake, and the filtrate was concentrated under reduced pressure to obtain 7.6g of a white solid in a yield of 91%, and TLC (DCM: MeOH ═ 12:1, v/v).

EXAMPLE 3 Synthesis of Compound 3

The compound 2(15g,96mmol) is added into a 250mL eggplant-shaped bottle, 150mL of phosphorus oxychloride is added, and the mixture is subjected to oil bath reflux reaction at 120 ℃ for 8 hours. After the reaction is finished, decompressing and concentrating to remove most of phosphorus oxychloride, dissolving the residue in 300mL of ethyl acetate, neutralizing the solution with 2M NaOH solution under the ice bath condition to be alkalescent, extracting, and sequentially using saturated NaHCO for an organic layer₃Washing the solution with saturated saline solution, drying with anhydrous sodium sulfate, vacuum filtering, concentrating the filtrate under reduced pressure to obtain yellow crude product, separating and purifying by column chromatography (PE: EA is 4:1) to obtain white solid 8.7g, yield 65%, TLC (P)E:EA＝2:1,v/v)。

EXAMPLE 4 Synthesis of Compound 4

Putting stannous chloride dihydrate into 150mL eggplant-shaped bottle₍45.13g,200mmol), 20mL of concentrated hydrochloric acid was added and dissolved, and Compound 3(5.52g,40mmol) was added under stirring at room temperature, and 45mL of ethyl acetate and N were added₂And (4) protecting, and carrying out reflux reaction at 78 ℃ for 0.5 h. After the reaction, the pH was adjusted to 7 with 6M NaOH solution in ice bath, followed by suction filtration, extraction of the filtrate with ethyl acetate, washing of the organic layer with saturated brine, drying over anhydrous sodium sulfate, suction filtration, and concentration of the filtrate under reduced pressure to obtain a yellow solid (3.8 g, yield 88%), TLC (PE: EA. RTM. 2:1, v/v).

EXAMPLE 5 Synthesis of Compound 5

125mL of distilled water and 50mL of DCM were added to a 500mL eggplant-shaped bottle, thiophosgene (2.04mL,26mmol) was added with thorough stirring, and compound 4(2.63g,24mmol) was dissolved in 75mL of DCM and then slowly added to the reaction solution, and after the addition was completed, stirring was carried out at room temperature for 1 hour. After the reaction, the mixture was allowed to stand for separation, and the organic phase was washed with distilled water and saturated brine three times in this order, dried over anhydrous sodium sulfate, suction-filtered, and the filtrate was concentrated under reduced pressure to obtain 1.78g of a yellow solid with a yield of 48%, TLC (PE: EA: 2:1, v/v).

EXAMPLE 6 Synthesis of Compound 6a

Adding 2-bromo-4' -methylacetophenone (4.2g,20mmol) into a 250mL eggplant-shaped bottle, dissolving with 100mL chloroform, adding hexamethylenetetramine (3.08g,22mmol), heating in an oil bath to 60 ℃, stirring for 5h, cooling to room temperature, filtering, suspending the collected precipitate in 100mL ethanol, adding 10mL concentrated hydrochloric acid, and heating in an oil bath to 85 ℃, and refluxing for 14 h. After the reaction was completed, suction filtration was performed, the filtrate was concentrated under reduced pressure, and the residue was recrystallized from methanol and ethyl acetate (1:50, v/v) to give 2.5g of a white solid in 85% yield, TLC (DCM: MeOH ═ 10:1, v/v).

EXAMPLE 7 Synthesis of Compound 6b

Adding 2-bromo-4' -fluoroacetophenone (4.3g,20mmol) into a 250mL eggplant-shaped bottle, dissolving with 100mL chloroform, adding hexamethylenetetramine (3.08g,22mmol), heating in an oil bath to 60 ℃, stirring for 5h, cooling to room temperature, filtering, suspending the collected precipitate in 100mL ethanol, adding 10mL concentrated hydrochloric acid, and heating in an oil bath to 85 ℃, and refluxing for 14 h. After the reaction was complete, suction filtration was performed, the filtrate was concentrated under reduced pressure, and the residue was recrystallized from methanol and ethyl acetate (1:50, v/v) to give 2.2g of a pale green solid in 73% yield, TLC (DCM: MeOH ═ 10:1, v/v).

EXAMPLE 8 Synthesis of Compound 6c

Adding 2,4' -dibromoacetophenone (5.5g,20mmol) into a 250mL eggplant-shaped bottle, dissolving with 100mL chloroform, adding hexamethylenetetramine (3.08g,22mmol), heating in an oil bath to 60 ℃, stirring for 5h, cooling to room temperature, filtering, suspending the collected precipitate in 100mL ethanol, adding 10mL concentrated hydrochloric acid, heating in an oil bath to 85 ℃, and refluxing for 14 h. After the reaction was completed, suction filtration was performed, the filtrate was concentrated under reduced pressure, and the residue was recrystallized from methanol and ethyl acetate (1:50, v/v) to give 2.9g of a pale yellow solid in 69% yield, TLC (DCM: MeOH ═ 10:1, v/v).

EXAMPLE 9 Synthesis of Compound 7a

Suspending a compound 5(0.69g,4.6mmol) and a compound 6a (0.75g,5.06mmol) in 50mL of DCM in a 150mL eggplant-shaped bottle, adding 9mL of 1M sodium carbonate solution, stirring at room temperature for 10min, heating the oil bath to 45 ℃ and stirring for 1h, then cooling and filtering, transferring a filter cake to a 100mL pressure-resistant tube, suspending the filter cake in 50mL of ethanol, heating the filter cake to 88 ℃ and refluxing for 12 h. After the reaction, the reaction mixture was cooled and filtered by suction to obtain 0.68g of a white solid in 52% yield, and TLC (PE: EA: 1, v/v).

EXAMPLE 10 Synthesis of Compound 7b

Suspending a compound 5(0.6g and 4mmol) and a compound 6b (0.83g and 4.4mmol) in 40mL of DCM in a 150mL eggplant-shaped bottle, adding 8mL of 1M sodium carbonate solution, stirring at room temperature for 10min, heating in an oil bath to 45 ℃, stirring for 1h, cooling, filtering, transferring a filter cake to a 100mL pressure-resistant tube, suspending in 40mL of ethanol, heating a module to 88 ℃, and refluxing for 12 h. After the reaction, the reaction mixture was cooled and filtered by suction to obtain 0.65g of a white solid in a yield of 57%, and TLC (PE: EA: 1, v/v).

EXAMPLE 11 Synthesis of Compound 7c

Suspending a compound 5(0.6g and 4mmol) and a compound 6c (0.93g and 4.4mmol) in 40mL of DCM in a 150mL eggplant-shaped bottle, adding 8mL of 1M sodium carbonate solution, stirring at room temperature for 10min, heating in an oil bath to 45 ℃, stirring for 1h, cooling, filtering, transferring a filter cake to a 100mL pressure-resistant tube, suspending in 40mL of ethanol, heating a module to 88 ℃, and refluxing for 12 h. After the reaction, the reaction mixture was cooled and filtered with suction to obtain 0.8g of a pale yellow solid in 58% yield, and TLC (PE: EA: 1, v/v).

EXAMPLE 12 Synthesis of Compound 8a

50mL eggplant-shaped bottleIn (1), compound 7a (0.37 g) was suspended in 8mL of POCl₃To the solution was added 0.8mL of DMF, N₂Protecting, heating the oil bath to 70 ℃, and stirring for 2 hours. After the reaction, phosphorus oxychloride was removed by concentration under reduced pressure, the residue was dissolved in 100mL of ethyl acetate, neutralized with saturated sodium bicarbonate solution under ice bath conditions to be weakly alkaline, extracted, the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a yellow crude product, which was subjected to column chromatography purification (PE: EA ═ 2:1) to obtain 0.2g of a white solid with a yield of 53%, and TLC (PE: EA ═ 1:1, v/v).

EXAMPLE 13 Synthesis of Compound 8b

In a 50mL eggplant-shaped bottle, Compound 7b (0.57g,2mmol) was suspended in 12mL of POCl₃To the solution was added 1.2mL of DMF, N₂Protecting, heating the oil bath to 70 ℃, and stirring for 2 hours. After the reaction, the reaction mixture was concentrated under reduced pressure to remove phosphorus oxychloride, the residue was dissolved in 150mL of ethyl acetate, neutralized with a saturated sodium bicarbonate solution under ice bath conditions to be weakly alkaline, extracted, the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a yellow crude product, which was subjected to column chromatography purification (PE: EA ═ 2:1) to obtain 0.35g of a pale yellow solid with a yield of 61%, and TLC (PE: EA ═ 1:1, v/v).

EXAMPLE 14 Synthesis of Compound 8c

In a 50mL eggplant-shaped bottle, Compound 7c (0.15g,0.4mmol) was suspended in 3mL of POCl₃To the solution was added 0.3mL of DMF, N₂Protecting, raising the temperature of the oil bath to 70 ℃, and stirring for 1 h. After the reaction is finished, concentrating under reduced pressure to remove phosphorus oxychloride, dissolving the residue in 50mL ethyl acetate, neutralizing the residue with saturated sodium bicarbonate solution under ice bath condition to be alkalescent, extracting, washing an organic layer with saturated saline solution, drying with anhydrous sodium sulfate, filtering, and concentrating the filtrate under reduced pressure to obtain yellow crude productThe product was purified by column chromatography (PE: EA ═ 2:1) to give an off-white solid 60mg, yield 40%, TLC (PE: EA ═ 1:1, v/v).

EXAMPLE 15 Synthesis of Compound M-1

A38 mL pressure bottle was charged with compound 8a (0.14g,0.5mmol) and p-anisidine (0.092g,0.75mmol), 5mL of n-butanol was added, and the mixture was heated to 130 ℃ with a heating block and stirred for 3 hours. After the reaction was completed, the reaction mixture was cooled, filtered, the filter cake was washed with distilled water with ultrasound for three times and then filtered again, and the filter cake was recrystallized from ethanol to obtain 85mg of a white solid with a yield of 46%, TLC (DCM: MeOH ═ 60:1, v/v). mp > 240 ℃;¹H NMR(400MHz,DMSO-d₆)δ9.03(s,1H),8.74(s,1H),7.98(s,1H),7.83(d,J＝8.1Hz,2H),7.63(d,J＝9.0Hz,2H),7.29(d,J＝8.0Hz,2H),6.96(d,J＝9.0Hz,2H),3.74(s,3H),2.33(s,3H)；¹³C NMR(100MHz,DMSO-d₆)δ156.00(s),142.87(s),140.53(s),137.79(s),132.59(s),130.92(s),130.06(s),128.57(s),126.01(s),125.74(s),123.74(s),114.44(s),107.50(s),55.77(s),21.36(s)；HR-MS(m/z)calculated for C₂₁H₁₈N₆O[M+H]⁺:371.1620,found:371.1612.

EXAMPLE 16 Synthesis of Compound M-2

The synthesis method is referred to example 15.

mp＞240℃；¹H NMR(400MHz,DMSO-d₆)δ9.45(s,1H),8.80(s,1H),8.11–8.08(m,2H),7.84(d,J＝8.1Hz,2H),7.78(dd,J₁＝8.0,J₂＝5.2Hz,1H),7.43(t,J＝9.1Hz,1H),7.29(d,J＝8.0Hz,2H),2.33(s,3H)；¹³C NMR(100MHz,DMSO-d₆)δ153.50(d,J＝242.2Hz),,142.14(s),139.44(s),138.17(s),137.19(s),131.49(s),130.10(s),125.85(s),122.42(s),121.38(d,J＝6.8Hz),119.52(d,J＝18.4Hz),117.19(s),108.00(s),21.41(s)；HR-MS(m/z)calculated for C₂₀H₁₄ClFN₆[M+H]⁺:393.1031,found:393.1024.

EXAMPLE 17 Synthesis of Compound M-3

A38 mL pressure bottle was charged with compound 8a (0.28mg,1mmol) and cyclopropylamine (0.2mL,2mmol), 10mL n-butanol was added, the temperature was raised to 130 ℃ with a heating block and stirred for 8 h. After the reaction, the reaction mixture was concentrated under reduced pressure, the residue was diluted with ethyl acetate, washed with saturated sodium bicarbonate solution and saturated brine in this order, the organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product in a brown-red color, which was separated and purified by column chromatography (PE: EA ═ 2:1) to obtain 0.16g of a white solid with a yield of 48%, and TLC (PE: EA ═ 1:1, v/v). mp > 240 ℃;¹H NMR(400MHz,DMSO-d₆)δ8.42(d,J＝16Hz,1H),8.12(s,1H),7.89(s,1H),7.76(d,J＝7.9Hz,2H),7.48(d,J＝20Hz,1H),7.26(d,J＝6.3Hz,2H),2.87(s,1H),2.31(s,3H),0.85–0.70(m,2H),0.67–0.53(m,2H)；¹³C NMR(100MHz,DMSO-d₆)δ143.60(s),137.74(s),133.53(s),132.74(s),125.80(s),120.69(s),107.23(s),106.78(s),24.75(s),21.37(s),6.93(s)；HR-MS(m/z)calculated for C₁₇H₁₆N₆[M+H]⁺:305.1515,found:305.1510.

EXAMPLE 18 Synthesis of Compound M-4

The synthesis method is referred to example 17.

mp＞240℃；¹H NMR(400MHz,DMSO-d₆)δ8.61(s,1H),8.13–7.86(m,1H),7.84–7.71(m,2H),7.36–7.15(m,2H),7.00(s,1H),4.36(s,1H),2.31(s,3H),2.13–1.87(m,2H),1.84–1.67(m,2H),1.67–1.41(m,4H)；¹³C NMR(100MHz,DMSO-d₆)δ142.56(s),137.53(s),132.61(s),131.23(s),129.96(s),125.67(s),120.35(s),107.12(s),53.48(s),32.74(s),24.15(s),21.37(s)；HR-MS(m/z)calculated for C₁₉H₂₀N₆[M+H]⁺:333.1828,found:333.1821.

EXAMPLE 19 Synthesis of Compound M-5

The synthesis method is referred to example 15.

mp＞240℃；¹H NMR(400MHz,DMSO-d₆)δ10.30(s,1H),9.23(s,1H),8.26(s,1H),8.24(s,2H),8.18(dd,J₁＝9.6,J₂＝2.2Hz,2H),7.85(d,J＝8.0Hz,2H),7.29(d,J＝8.0Hz,2H),2.32(s,3H)；¹³C NMR(100MHz,DMSO-d₆)δ146.57(s),141.99(s),138.70(s),138.48(s),138.30(s),131.64(s),130.15(s),128.64(s),125.96(s),125.29(s),119.73(s),109.39(s),21.43(s)；HR-MS(m/z)calculated for C₂₀H₁₅N₇O₂[M+H]⁺:386.1365,found:386.1359.

EXAMPLE 20 Synthesis of Compound F-1

The synthesis method is referred to example 15.

mp＞240℃；¹H NMR(400MHz,DMSO-d₆)δ9.04(s,1H),8.76(s,1H),7.98–7.94(m,2H),7.64–7.61(m,2H),7.32(t,J＝8.9Hz,2H),6.97–6.95(m,2H),3.74(s,3H)；¹³C NMR(100MHz,DMSO-d₆)δ162.39(d,J＝244.5Hz),156.01(s),142.03(s),140.40(s),132.57(s),130.43(d,J＝2.8Hz),127.77(d,J＝8.0Hz),123.77(s),116.43(d,J＝21.6Hz),114.43(s),107.82(s),55.76(s),48.96(s)；HR-MS(m/z)calculated for C₂₀H₁₅FN₆O[M+H]⁺:375.1370,found:375.1364.

EXAMPLE 21 Synthesis of Compound F-2

The synthesis method is referred to example 15.

mp＞240℃；¹H NMR(400MHz,DMSO-d₆)δ9.59(s,1H),8.96(s,1H),8.17–8.09(m,2H),8.02–7.91(m,2H),7.80(ddd,J₁＝9.0Hz,J₂＝4.2Hz,J₃＝2.7Hz,1H),7.56–7.35(m,2H),7.35–7.26(m,2H)；¹³C NMR(100MHz,DMSO-d₆)δ162.53(d,J＝244.9Hz),153.51(d,J＝242.1Hz),141.02(s),139.47(s),138.91(s),137.17(d,J＝2.2Hz),131.51(s),129.50(s),128.62(s),127.91(d,J＝8.3Hz),121.42(d,J＝6.8Hz),119.52(d,J＝18.4Hz),117.29(d,J＝21.8Hz),116.51(d,J＝21.6Hz),108.55(s)；HR-MS(m/z)calculated for C₁₉H₁₁ClF₂N₆[M+H]⁺:397.0780,found:397.0774.

EXAMPLE 22 Synthesis of Compound F-3

The synthesis method is referred to example 17.

mp＞240℃；¹H NMR(400MHz,DMSO-d₆)δ8.44(d,J＝19.2Hz,1H),8.12(s,1H),7.91(s,1H),7.88(d,J＝8.6Hz,2H),7.48(d,J＝19.9Hz,1H),7.29(dd,J₁＝15.8,J₂＝8.7Hz,2H),2.93–2.81(m,1H),0.84–0.73(m,2H),0.66–0.56(m,2H)；¹³C NMR(100MHz,DMSO-d6)δ162.35(d,J＝244.4Hz),143.59(s),133.61(s),132.74(s),130.49(d,J＝14.6Hz),127.72(d,J＝8.0Hz),120.73(s),116.42(d,J＝21.4Hz),107.61(s),107.12(s),24.75(s),6.94(s)；HR-MS(m/z)calculated for C₁₆H₁₃FN₆[M+H]⁺:309.1264,found:309.1256.

EXAMPLE 23 Synthesis of Compound F-4

The synthesis method is referred to example 17.

mp＞240℃；¹H NMR(400MHz,DMSO-d₆)δ8.69–8.54(m,1H),8.05(d,J＝1.6Hz,1H),7.91(dd,J₁＝8.2Hz,J₂＝5.7Hz,2H),7.83(d,J＝1.5Hz,1H),7.38–7.22(m,2H),6.99(dd,J₁＝20.7Hz,J₂＝6.5Hz,1H),4.45–4.27(m,1H),2.11–1.94(m,2H),1.81–1.69(m,2H),1.66–1.49(m,4H)；¹³C NMR(100MHz,DMSO-d₆)δ162.34(d,J＝244.6Hz),142.69(s),141.90(s),129.97(s),127.75(d,J＝7.8Hz),116.30(d,J＝21.6Hz),107.59(s),53.50(s),32.62(s),24.01(s)；HR-MS(m/z)calculated for C₁₈H₁₇FN₆[M+H]⁺:337.1577,found:337.1570.

EXAMPLE 24 Synthesis of Compound F-5

The synthesis method is referred to example 15.

mp＞240℃；¹H NMR(400MHz,DMSO-d₆)¹H NMR(400MHz,DMSO-d6)δ9.93(s,1H),8.85(s,1H),8.31–8.22(m,2H),8.18(s,1H),8.06(d,J＝7.9Hz,2H),7.99(d,J＝5.5Hz,2H),7.33(t,J＝7.7Hz,2H)；HR-MS(m/z)calculated for C₁₉H₁₂FN₇O₂[M+H]⁺:390.1115,found:390.1108.

EXAMPLE 25 Synthesis of Compound B-1

The synthesis method is referred to example 15.

mp＞240℃；¹H NMR(400MHz,DMSO-d₆)δ9.65(s,1H),9.26(s,1H),8.07(s,1H),7.88(d,J＝8.5Hz,2H),7.76–7.63(m,4H),6.97(d,J＝9.0Hz,2H),3.74(s,3H)；¹³C NMR(100MHz,DMSO-d₆)δ156.43(s),140.67(s),138.94(s),132.59(s),131.85(s),131.50(s),131.25(s),129.10(s),127.87(s),124.19(s),122.05(s),114.52(s),110.02(s),55.81(s)；HR-MS(m/z)calculated for C₂₀H₁₅BrN₆O[M+H]⁺:435.0569,found:435.0564.

EXAMPLE 26 Synthesis of Compound B-2

The synthesis method is referred to example 15.

mp＞240℃；¹H NMR(400MHz,DMSO-d₆)δ9.49(s,1H),8.94(s,1H),8.11–8.07(m,2H),7.89(s,1H),7.87(s,1H),7.81–7.74(m,1H),7.68(d,J＝8.4Hz,2H),7.43(t,J＝9.1Hz,1H)；¹³C NMR(100MHz,DMSO-d₆)δ153.51(d,J＝246.3Hz),140.86(s),139.38(s),138.99(s),137.15(d,J＝2.9Hz),132.46(s),132.26(s),131.49(s),127.79(s),122.38(s),121.66(s),121.33(d,J＝6.8Hz),119.51(d,J＝18.5Hz),117.25(d,J＝21.7Hz),109.14(s)；HR-MS(m/z)calculated for C₁₉H₁₁BrClFN₆[M+H]⁺:456.9979,found:456.9974.

EXAMPLE 27 Synthesis of Compound B-3

The synthesis method is referred to example 17.

mp＞240℃；¹H NMR(400MHz,DMSO-d₆)δ8.52(d,J＝16.5Hz,1H),8.12(s,1H),7.89(s,1H),7.86–7.76(m,2H),7.72–7.58(m,2H),7.48(d,J＝20Hz,1H),2.96–2.78(m,1H),0.86–0.69(m,2H),0.67–0.55(m,2H)；¹³C NMR(100MHz,DMSO-d₆)δ143.57(s),133.19(s),132.42(s),127.67(s),121.24(s),108.25(s),107.80(s),24.76(s),6.93(s)；HR-MS(m/z)calculated for C₁₆H₁₃BrN₆[M+H]⁺:369.0463,found:369.0460.

EXAMPLE 28 Synthesis of Compound B-4

The synthesis method is referred to example 17.

mp＞240℃；¹H NMR(400MHz,DMSO-d₆)δ8.69(s,1H),7.94(s,1H),7.86–7.80(m,2H),7.70–7.63(m,2H),7.01(d,J＝6.4Hz,1H),4.50–4.23(m,1H),2.07–1.95(m,2H),1.79–1.68(m,2H),1.64–1.54(m,4H)；¹³C NMR(100MHz,DMSO-d₆)δ142.51(s),141.59(s),133.23(s),132.41(s),127.67(s),121.19(s),108.24(s),53.51(s),32.73(s),24.14(s)；HR-MS(m/z)calculated for C₁₈H₁₇BrN₆[M+H]⁺:397.0776,found:397.0773.

Example 29 the following are some of the pharmacological tests and results of representative compounds of the invention:

1. detecting the Effect of Compounds on the in vitro proliferation of human tumor cells

1.1 Experimental methods

The experimental method comprises the following steps:

1) digesting and counting cells, preparing 3.5 multiplied by 104 cell suspensions/ml, and adding 100 mul of cell suspensions into each hole of a 96-hole cell culture plate;

2) the 96-well cell culture plate was placed at 37 ℃ and 5% CO₂Culturing in an incubator for 24 hours;

3) diluting the drug with culture medium to required working solution concentration, adding 100 μ l corresponding drug-containing culture medium into each well, and setting up negative control group;

4) the 96-well cell culture plate was placed at 37 ℃ and 5% CO₂Culturing for 72 hours in an incubator;

5) a 96-well plate is subjected to CCK-8 staining, and the OD value is measured when the lambda is 450 nm;

a) adding 10 mul CCK-8 into each hole, and continuously culturing for 2-3 hours in an incubator;

b) mixing the mixture gently by a shaking table for 10 minutes to remove bubbles in the 96-well plate;

c) and lambda is 450nm, and the OD value of each hole is read by a microplate reader to calculate the inhibition rate.

6) And calculating the inhibition rate of each group.

Sample concentration: 10 μ M. Half inhibitory concentration IC50 was calculated from the inhibition rate using the LOGIT method. The experiment was repeated 3 more times and the data are expressed as Mean ± SD.

1.2 data processing

All data are expressed as means ± standard deviation (Mean ± SD) and statistical analysis is performed using SPSS 16.0 software. Multiple comparisons between groups were performed using a completely randomly designed one-way ANOVA. Assuming that the test level is judged as α ═ 0.05, P <0.05 indicates that the difference is statistically significant.

1.3 results and discussion

In the experiment for detecting the influence of compounds on the in vitro proliferation of human tumor cells, two cells, namely human chronic myelogenous leukemia cell K562 and human colon cancer cell HCT116, are selected for activity detection, cancer cells growing in logarithmic phase are connected into a 96-well cell culture plate, 100 mu L of cell suspension is added into each well, after the cells are cultured for 24h, the cells are administrated, the administration concentration is set to be 10 mu M, after the drugs act for 72h, CCK-8 staining is carried out, the OD value is measured, and the inhibition rate is calculated. The results are shown in Table 1.

TABLE 1 Single concentration inhibition of K562 and HCT116 by the target Compounds

As can be seen from Table 1: the compound shows certain selectivity on the proliferation inhibition of K562 tumor cells and HCT116 tumor cells under the condition of 10 mu M, the inhibition activity on HCT116 cells is mostly not ideal, the difference with a positive control drug is larger, and the inhibition activity on K562 cell strains is relatively better. Compared with the positive control drug AT9283, the compound M-2 has obvious inhibition effect on K562 cells, and the inhibition rate AT the concentration of 10 mu M is 83.94 percent and exceeds the positive control drug AT9283(80.53 percent). The compounds M-5, F-1, F-2 and B-2 also showed better inhibitory activity to K562 cells, and the inhibition rates at 10 μ M concentration were 61.34%, 60.43%, 48.33% and 58.97%, respectively.

Selecting compounds M-2, M-5, F-1, F-2 and B-2 with high single-concentration inhibition rate on K562 cells to determine the half Inhibition Concentration (IC) of the compounds on the growth of tumor cells₅₀) 3 duplicate wells were set for each drug concentration and the test results are shown in table 2.

Inhibitory Activity of the Compounds of Table 2 on K562

As can be seen from Table 2: the tested compound has better cell proliferation inhibition activity on K562 cells, and IC thereof₅₀The value is in the range of 5.597-11.724. mu.M, wherein the inhibitory activity of compound M-2 on K562 cells is best, IC₅₀5.597 ± 0.020 μ M. The inhibitory activity of the compound B-2 is also considerable, IC₅₀＝8.081±0.010μM。

2. Method for detecting apoptosis by Annexin-V FITC/PI double staining method

1) Digesting and inoculating the cells in logarithmic growth phase into a six-well plate, adding corresponding drug-containing culture medium according to the group setting after the cells adhere to the wall, and simultaneously setting up a negative control group (without adding drugs, only adding DMSO); after 72h of drug action, the cells were harvested by digestion with 0.25% pancreatin (without EDTA); wash cells twice with PBS (centrifuge 1000rpm, 5min) to harvest 5X 10⁵A cell;

2) adding 500 mu L Binding Buffer suspension cells;

3) adding 5 mu L Annexin V-FITC, mixing, adding 5 mu L Propidium Iodide, and mixing;

4) reacting at room temperature in dark for 5-15 min; detecting the apoptosis condition by using a flow cytometer.

The results are shown in Table 3.

TABLE 3

As can be seen from Table 3: compound M-2 induced apoptosis at 10. mu. mmol. Other compounds such as M-1, M-5, F-1, F-2, B-2, etc. have similar experimental effects.

3. PI single staining method for detecting cell cycle

1) Digesting and inoculating the cells in the logarithmic growth phase into a six-hole plate, and adding the drugs according to the groups after the cells adhere to the wall the next day;

2) after 72h of action, the cells were harvested by digestion with 0.25% pancreatin (without EDTA);

3) washing the cells twice with PBS (centrifugation 1000rpm, 5min) to collect 5X 105 cells;

4) fixing the prepared single cell suspension with 70% ethanol for 2 hr (or overnight), storing at 4 deg.C, washing the fixing solution with PBS before staining (if necessary, filtering the cell suspension with 200 mesh sieve once);

5) adding 100 μ L RNase A in 37 deg.C water bath for 30 min;

6) adding 400 μ L PI, dyeing, and mixing, keeping in dark at 4 deg.C for 30 min;

7) and (5) detecting by using a computer, and recording red fluorescence at the 488nm excitation wavelength.

The results are shown in Table 4.

TABLE 4

As can be seen from Table 4: as the concentration of compound M-2 increases, cell mitosis is arrested in S phase. Other compounds such as M-1, M-5, F-1, F-2, B-2, etc. have similar experimental effects.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A compound represented by formula I or a pharmaceutically acceptable salt thereof:

wherein the content of the first and second substances,

R²represents phenyl, substituted phenyl, benzyl, substituted benzyl, C₁-C₆Alkyl, substituted C₁-C₆Alkyl, cyclopropyl, substituted cyclopropyl, cyclobutyl, substituted cyclobutyl, cyclopentyl, substituted cyclopentyl, cyclohexyl or substituted cyclohexyl, said substituted phenyl, substituted benzyl, substituted C₁-C₆The alkyl, substituted cyclopropyl, substituted cyclobutyl, substituted cyclopentyl or substituted cyclohexyl group may be optionally mono-or polysubstituted with the following substituents: c₁-C₄Alkoxy, cyano, hydroxy, nitro or halogen.

2. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R¹Represents C₁-C₄Alkyl, substituted C₁-C₄Alkyl radical, C₁-C₄Alkoxy, cyano, hydroxy, nitro, fluoro or bromo, said substituted C₁-C₄The alkyl group may be optionally mono-or polysubstituted with the following substituents: a hydroxyl group,Nitro, fluoro, chloro or bromo.

3. A compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein R¹Represents methyl, ethyl, methoxy, ethoxy, fluorine, chlorine or bromine.

4. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R²Represents phenyl, substituted phenyl, cyclopropyl, substituted cyclopropyl, cyclopentyl or substituted cyclopentyl, which may be optionally mono-or polysubstituted with: methoxy, ethoxy, cyano, hydroxy, nitro or fluoro, chloro or bromo.

5. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R²Represents phenyl, substituted phenyl, cyclopropyl, cyclopentyl, said substituted phenyl being optionally mono-or polysubstituted with the following substituents: methoxy, ethoxy, cyano, hydroxy, nitro, fluoro, chloro or bromo.

6. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R²Represents phenyl or substituted phenyl, said substituted phenyl being optionally mono-or polysubstituted with the following substituents: methoxy, ethoxy, nitro, fluoro, chloro or bromo.

7. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

8. the compound according to claim 7, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

9. a process for the preparation of a compound according to claim 1,

10. a pharmaceutical composition comprising a compound according to any one of claims 1, 2, 3, 4, 5, 6, 7 or 8 or a pharmaceutically acceptable salt thereof as an active ingredient or as a major active ingredient, together with a pharmaceutically acceptable carrier.

11. Use of a compound according to any one of claims 1, 2, 3, 4, 5, 6, 7 or 8, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disease associated with a tumour.